125 results about "Selectable marker" patented technology

Provided herein are transposon / transposase systems for transposing and selecting of transposed cells where the system includes a transposase or polynucleotide encoding a transposase; and an exogenous nucleic acid sequence comprising: a transposon comprising: a 5' inverted terminal repeat (ITR), a first polynucleotide encoding a gene of interest; and a 3' inverted terminal repeat (ITR); and (ii) a polynucleotide encoding an at least first selection marker located outside of the 5' ITR and 3' ITR, where upon introducing (a) and (b) into a cell the transposon is transposed into the cell's genome and the polynucleotide encoding the selection marker is episomal.

Transgene-free genome editing of plants in T0 generation is highly desirable but challenging, especially in perennials and vegetatively propagated plants. Here is provided a co-editing strategy for generating transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE) / gRNA-Cas 12a / crRNA-GFP in plants. Specifically, CBE / gRNA was used to base edit the AES gene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Casl2a / crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene- free genome-edited plants were efficiently generated for various genes (either individual or multiplex) in tomato, tobacco, potato, and citrus in the T0 generation. The biallelic / homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 8% to 50%. Whole genome sequencing further confirmed transgene-free and absence of off- target mutations in the edited plants. The co-editing strategy is efficient for generating transgene- free, genome-edited plants in the T0 generation, thus being a potent tool for plant genetic improvement.

A method of producing a population of mature human pluripotent stem cell-derived sensory neurons (hPSC-SNs) expressing a target gene associated with at least one of nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition, the method comprising introducing into a population of human pluripotent stem cells (hPSCs) a composition comprising at least one site-directed nuclease targeting a site within the target gene, and at least one nucleic acid comprising a nucleotide sequence encoding at least one screenable, selectable marker that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene, wherein the target site is located downstream of the open reading frame of the target gene, and wherein the site-directed nuclease cleaves the target site of the target gene and the nucleic acid encoding the screenable, selectable marker is inserted at the target site; covering said population of hPSCs under an extracellular matrix comprising at least one neuronal differentiation driver to produce a sensory committed neural crest population; contacting said sensory committed neural crest population with at least one neuronal differentiation driver and at least one neurotrophic factor to produce a population of early sensory neurons (SNs); and contacting said population of early SNs with at least one neurotrophic factor to produce a population of mature hPSC-SNs expressing at least one of SN marker or one pan neuronal marker; isolating the cells expressing the at least one screenable, selectable marker, wherein said population of mature hPSC-SNs expressed said one target gene associated with nociceptive pain, chronic pain, pruriception a nociceptive- or pruriceptive- mediated condition, and wherein said population of mature hPSC-SNs responds to a nociceptive and pruriceptive stimulus.

The invention provides application of a pig STR molecular marker combination in pig individual identification, a kit and an identification method, relates to the technical field of biology, and develops an STR molecular marker combination for accurate identification of pig individual identification by applying a whole genome association analysis method for 18 wild boar varieties and 42 domestic pig varieties. By detecting the combination, individual identification and genetic identification can be accurately and efficiently carried out on a to-be-detected pig sample, and when the minimum set number of the selected STR marker is 6, individual distinguishing can be carried out. According to the scheme provided by the invention, by comprehensively analyzing the STR typing result, the parent-child margin determination and individual identification of the pig can be effectively carried out, so that the pedigree accuracy in pig breeding work can be improved, the breeding process is accelerated, and the method has good application prospects and economic values.

The present invention relates to a recombinant vector for expression of a target protein or target gene containing a glutamine synthetase split fragment, and a method for selection of a cell line expressing high levels of a recombinant protein using same. According to the present invention, the efficiency of selecting cell lines for biopharmaceutical production dramatically increases,, and it can be used free from stability problems because it is an improvement on the conventionally used selectable marker GS (glutamine synthetase).

The present invention relates generally to the field of molecular biology and concerns a method for high throughput screening and characterization of one or more candidate plant pathogen effect genes and / or one or more candidate plant disease resistance genes in a single plant cell, the method comprises: (i) introducing into a plant protoplast one or more expression cassettes comprising at least one candidate plant pathogen effect gene and / or at least one candidate plant disease resistance gene; (ii) transiently expressing the one or more candidate plant pathogen effect genes and / or the one or more candidate plant disease resistance genes; (iii) measuring reactive oxygen species (ROS) produced by the protoplast; then (iv) separating the plant protoplast producing the ROS; and (v) identifying the introduced at least one candidate plant pathogen effect gene and / or at least one candidate plant disease resistance gene. Alternatively, the protoplast is capable of expressing the selectable marker, thereby allowing for the measurement of modulated selectable marker expression as an alternative indicator for HR activation.

Recombinant NK cells, and particularly recombinant NK-92 cells express an anti-B7-H4 chimeric antigen receptor (CAR) having an intracellular domain of FcεRIγ. Most notably, CAR constructs with an intracellular domain of FcεRIγ had a significantly extended duration of expression and cytotoxicity over time. The anti-B7-H4 CAR may be expressed from RNA and DNA, preferably from a tricistronic construct that further encodes CD16 and a cytokine to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of the recombinant proteins and provide a convenient selection marker to facilitate rapid production of recombinant NK / NK-92 cells.

Disclosed is an expression vector comprising a polynucleotide encoding for a glutamine synthetase with reduced activity compared to a wild type glutamine synthetase. Also disclosed are host cells, methods for preparing stable cell line, methods of producing polypeptide of interest, and kits thereof.

The present invention provides a novel expression system comprising a mammalian selectable marker that promotes the desired post-translational modification of a glycoprotein. Specifically, the present invention includes methods and compositions for achieving optimal recombinant protein expression in mammalian cells by using a selectable marker system based on a GPT gene of mammalian origin. The present invention includes methods for promoting the selectivity of recombinant proteins and increasing expression copies and protein yields in mammalian cells, and methods for using the GPT expression system.

A method of producing a population of mature human pluripotent stem cell-derived sensory neurons (hPSC-SNs) expressing a target gene associated with at least one of nociceptive pain, chronic pain, pruriception and a nociceptive- or pruriceptive- mediated condition, the method comprising introducing into a population of human pluripotent stem cells (hPSCs) a composition comprising at least one site-directed nuclease targeting a site within the target gene, and at least one nucleic acid comprising a nucleotide sequence encoding at least one screenable, selectable marker that is flanked by (i) a nucleotide sequence homologous with a region located upstream of the target site within the target gene and (ii) a nucleotide sequence homologous with a region located downstream of the target site within the target gene, wherein the target site is located downstream of the open reading frame of the target gene, and wherein the site-directed nuclease cleaves the target site of the target gene and the nucleic acid encoding the screenable, selectable marker is inserted at the target site; covering said population of hPSCs under an extracellular matrix comprising at least one neuronal differentiation driver to produce a sensory committed neural crest population; contacting said sensory committed neural crest population with at least one neuronal differentiation driver and at least one neurotrophic factor to produce a population of early sensory neurons (SNs); and contacting said population of early SNs with at least one neurotrophic factor to produce a population of mature hPSC-SNs expressing at least one of SN marker or one pan neuronal marker; isolating the cells expressing the at least one screenable, selectable marker, wherein said population of mature hPSC-SNs expressed said one target gene associated with nociceptive pain, chronic pain, pruriception a nociceptive- or pruriceptive- mediated condition, and wherein said population of mature hPSC-SNs responds to a nociceptive and pruriceptive stimulus.

Levels of expression of antibiotic resistance genes are increased up to six-fold by inserting a proteasome-targeting tag into transgenes expressed in eukaryotic cells. Various selectable marker proteins are combined with different destabilization domains, leading to up to 70% increase in transgene expression. The increase in expression varies highly depending on the engineered construct and the lines cells used. Increase in expression drives exosome loading of cargo proteins in some aspects. By increasing expression and by editing trafficking signals of cargo proteins, proteins that normally locate to the ER can be trafficked to exosomes. This disclosure discloses efficient exosome delivery of a wide variety of engineered proteins, including modified antigen proteins of SARS-CoV-2 and influenza, and other proteins such as a modified alpha galactosidase A, an extracellular domain of vascular endothelial growth factor fused to a constant region of a human immunoglobulin heavy chain, and modified trastuzumab heavy and light chains.

The invention provides methods of using chloroplast-selectable markers to detect nuclear genome editing. The methods involve selecting a genetic modification that has occurred in a nuclear genome of a photosynthetic cell; transforming a photosynthetic cell with a ribonucleoprotein complex that effects a genetic modification in the genome in the nucleus of a photosynthetic cell; transforming the photosynthetic cell with a construct that effects an insertion of a selectable marker into the plastome of a plastid of the cell; selecting for photosynthetic cells having the selectable marker inserted into the plastome; and thereby selecting for the genetic modification to the genome in the nucleus of the photosynthetic cell.

Dual vector systems and these comprehensive mammalian host cells are provided for the expression of multi-chain molecules, with each vector employing a different promoter controlling the expression of a selectable marker. The mammalian host cells can be used to generate a recombinant protein of interest in a mammalian cell culture.

The present invention relates to a strain of filamentous fungus belonging to the Ascomycetes division, excluding fungi belonging to the Saccharomycetes class, in which a gene encoding an arginine permease (CAN1), or a variant or orthologous gene, has been inactivated. The invention also relates to the various uses of this strain, as well as the genetic modification process for obtaining a strain according to the invention. The invention further relates to the use of L-canavanine as a selection marker for transformants in which a gene encoding an arginine permease (CAN1), or a variant or orthologous gene, has been inactivated. (no figure)

Dual vector systems and mammalian host cells comprising these dual vector systems are provided, wherein each vector employs a different promoter that drives the expression of a selectable marker. A mammalian host cell can be used to produce a recombinant protein of interest in mammalian cell culture.

The invention provides a method for dynamically regulating and controlling the copy number of plasmids in saccharomyces cerevisiae based on RNA (Ribonucleic Acid) interference. According to the method, an orthogonal RNAi system is reconstructed, saccharomyces cerevisiae is used as a synthetic biological chassis with a dynamic programmable plasmid copy number, a heterologous RNAi mechanism from Ckstri saccharomyces cerevisiae is integrated, sequence-specific siRNA of a targeted plasmid coding selective marker is designed, a chemically induced gene dose control platform is established, and the gene dose control platform is used for controlling the gene dose. The dynamic regulation and control of the plasmid copy number are realized, so that the gene expression dosage is optimized, and the biosynthesis efficiency is improved. RNAi-mediated copy number regulation is applied to a carotenoid biosynthesis pathway, and compared with a static plasmid system, the lycopene titer is increased by 18.6 times. The method further enriches an anabolism regulation tool set, has huge potential in the aspect of improving the production performance of a microbial cell factory, and provides a new thought for metabolic engineering optimization in the field of synthetic biology.

Herein is reported a nucleic acid comprising in operably linked form a nucleic acid encoding a selection marker, a nucleic acid encoding a self-cleaving peptide sequence, and a nucleic acid encoding a proteinaceous protease inhibitor. Further reported are methods for the recombinant production of a heterologous polypeptide using said nucleic acid as well as a cell comprising said nucleic acid according to the invention. Likewise reported is the use of the nucleic acid according to the invention for increasing the amount of the recombinantly produced heterologous polypeptide by reducing protease cleavage.

Provided are methods for inserting a large gene / polynucleotide between about 4.5Kb and about 8Kb and lacking a selection marker into a target genomic position in a cell by delivering the gene / polynucleotide by two AAV vectors in the presence of a p53-binding protein 1 (53BP1) inhibitor and a DNA-dependent protein kinase catalytic subunit (DNA- PKcs) inhibitor. The disclosed methods improve large gene insertion by at least 10-fold and obviate the need for a selection marker in the vector design, thereby providing more room in the AAV vector for gene delivery.

The present invention is within the field of industrial protein production. The invention provides a novel expression system using dehydroorotate dehydrogenase (DHODH) as a selection marker in combination with leflunomide or a metabolite thereof, notably for use in mammalian cell lines. Expression vectors encoding DHODH, cell lines comprising said vectors and methods of producing recombinant proteins are also provided.

The application discloses a dCAPS molecular marker for detecting a residual amount of a cucumber fruit myclobutanil and application thereof, and relates to the technical field of molecular markers.The dCAPS molecular marker is derived from a SNP at a position of 158 of a CsABCI19 gene promoter.The dCAPS molecular marker is applied to detection of a residual amount of a cucumber fruit myclobutanil.The method comprises the following steps: (1) extracting DNA; (2) performing a PCR reaction; and (3) performing Nco I enzyme cutting, so that two band types are obtained, one is 491 bp, corresponding to SNP A, and the other is 444 bp and 48 bp, corresponding to SNP G, and the two types are identified as a fruit low myclobutanil residual amount germplasm and a fruit high myclobutanil residual amount germplasm respectively.The verification result of the dCAPS molecular marker in the germplasm resource shows that the identification result of the molecular marker is consistent with the phenotype, and the dCAPS molecular marker can be used for rapidly identifying the cucumber germplasm resource with a low myclobutanil residual amount in a fruit, and is a good breeding auxiliary selection marker.

The present invention relates to a method for integrating one or more recombinant sites in a sole target gene of a cell genome comprising carrying out sequentially multiple site-specific recombination by using alternatively two different serine-integrases, wherein the selection marker is removed after each site-specific recombination by different transposases: An in vitro method for inserting at least one recombinant site in a target gene / locus from an isolated cell genome, said method comprising integrating a nucleotide sequence A into the target gene from an isolated cell genome by homologous recombination, wherein said nucleotide sequence A comprises a recombination cassette, flanked at 5′ and 3′ by target gene homology arms, and removing the selection marker by using the second transposase, thus obtaining a cell genome A comprising (i), (ii) and (iii) of nucleotide sequence A.

The invention discloses an SNP molecular marker related to cottonseed oil content and application of the SNP molecular marker, and belongs to the technical field of molecular marker development. The SNP molecular marker is located at the 43284845th site of an upland cotton genome D03 chromosome, the nucleotide is A or G, the upstream and downstream sequences are shown as SEQ ID NO.1, and the G allele is associated with the high oil content character. The SNP molecular marker can be directly applied to (1) identification and screening of high-oil cotton germplasm resources: high-oil materials are rapidly screened from existing varieties or strains; (2) cotton molecular breeding practice: as an effective selection marker, the cotton molecular marker is used for guiding the breeding of filial generations, and by selecting individuals carrying G alleles (especially GG type), the directionality and success rate of breeding new varieties / lines of high-oil cotton are remarkably improved. The method has very important practical application value.

Recombinant NK cells, and particularly recombinant NK-92 cells express an anti-B7-H4 chimeric antigen receptor (CAR) having an intracellular domain of FcεRIγ. Most notably, CAR constructs with an intracellular domain of FcεRIγ had a significantly extended duration of expression and cytotoxicity over time. The anti-B7-H4 CAR may be expressed from RNA and DNA, preferably from a tricistronic construct that further encodes CD16 and a cytokine to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of the recombinant proteins and provide a convenient selection marker to facilitate rapid production of recombinant NK / NK-92 cells.

Disclosed by the present application are an antibody library construction method and an application thereof. The method comprises the following steps: inserting a first element and a second element into a same vector or different vectors, and transfecting the vectors into the cells to obtain an antibody expression cell library, i.e., the antibody library. The first element comprises CIS activators and selection marker genes; the second element comprises extracellular antibody library coding domain, Notch nuclear structure domain and intracellular transcription structure domain.

The present invention relates to vectors and their use in developing host cell lines for producing proteins of interest, and in particular to vectors utilizing weak promoters to drive selectable markers.

The present invention relates to a method for editing the genome of a cell, such as a yeast cell. The method of the present invention requires the cell to be contacted with at least one ribonucleoprotein, at least one donor-DNA construct and a selectable marker such that they are introduced into the cell. The present invention is especially suitable for multiplex genome editing of cells such as yeast cells. The current invention further relates to a composition, a cell obtainable by the method of the invention and a method for the production of a compound of interest.

The disclosure provides, for example, a method of making a composition comprising purified DNA molecules. In some embodiments, the method comprises contacting a solution comprising a plurality of circular, double-stranded DNA molecules to a hydrophobic interaction chromatography matrix. In some embodiments, the circular, double-stranded DNA molecules lack one or both of a bacterial origin of replication and a selectable marker.

The present invention provides for the production of improved viral and non-viral nanoplasmid vectors. Specifically, the invention provides an antibiotic-label-free covalent closed circular recombinant DNA molecule comprising: a. An antibiotic-label-free insert comprising a structured DNA sequence selected from the group consisting of a reverse repeat, a forward repeat, a homopolymerized repeat and an eukaryotic origin of replication; b. A Pol III-dependent origin of replication comprising an R6K [gamma] origin of replication having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 18; and c. An RNA-OUT RNA selection marker, which comprises an RNA-IN modulating RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6, and a method for detecting the RNA-OUT RNA.

The invention provides a DNA marker plasmid as well as a combination system and application thereof, and relates to the field of molecular biology. The DNA marker plasmid is formed by connecting a linearized skeleton sequence and an insertion fragment sequence; the linearized skeleton sequence is derived from a reference plasmid from which a lactose operon sequence is deleted, and a selectable marker gene of the linearized skeleton sequence has a preset recognition site of restriction enzyme; the insertion fragment sequence is formed by connecting a plurality of DNA fragments in series, and recognition sites of restriction endonuclease are arranged between the adjacent DNA fragments. According to the plasmid, a restriction enzyme cutting site is introduced into a selective marker, so that a skeleton is directly used as a characteristic band, and the complete utilization of a sequence is realized. Waste and purification are avoided after enzyme digestion, the cost is remarkably reduced, and the preparation efficiency is improved.